Project Summary Chlamydia trachomatis has an immense impact on public health in the US and worldwide. Despite this impact, there is a paucity of virulence factors that have experimentally defined and evaluated. This is largely due to the very recent development of genetic tools and capabilities. One tool that has not been developed yet is transposon mutagenesis. This is a very powerful approach to generate single gene deletions and evaluate influence on specific phenotype. As our preliminary data report, we have demonstrated that the himar transposon system is functional and effective in generating transposon insertion mutant strains of Chlamydia. This proposal is designed to expand and generate a defined transposon library of Chlamydia trachomatis (Specific Aim 1). Due to the obligate intracellular nature of Chlamydia, disruptive transposon insertions support that these gene products are not essential for in vitro (tissue culture) growth. Given the highly evolved and condensed genetic repertoire, we hypothesize that some of these unessential in vitro genes are important for in vivo (vertebrate mammal) infection. To test this hypothesis, we are evaluating the relative bacterial burden of mutant strains in a mouse model. Through these efforts it is expect to experimentally discover novel virulence factors encoded by Chlamydia trachomatis. These discoveries may lead to new treatment or prevention strategies for Chlamydia or other microbial infectious diseases.